(a) Field of the Invention
The present invention relates to an epitope of HN protein in Newcastle disease virus which can be recognized by an avian immune system and an antibody against the epitope, a method for detecting a Newcastle disease virus by using the antibody, an antigenic variant of Newcastle disease virus carrying changes in the epitope, and a vaccine prepared by using the antigenic variant.
(b) Description of the Related Art
Newcastle disease is very infectious and lethal in fowls and thus is designated as the first communicable disease by law in Korea. If an unimmunized chicken is infected, the mortality is 100%. An infection to Egg-laying hen which is not properly immunized shows a respiratory or a digestive disorder, and reduction of laying eggs, thereby doing economic damage on poultry man. Although the occurrence of Newcastle disease is notified annually, it increases continuously and is spread all over the nation. In addition, the chicken which has a low titer of antibody in spite of immunization shows nervous system disorder such as torticolis.
Newcastle disease virus is a single stranded RNA virus belonging to genus Avulavirus. The envelope of Newcastle disease virus includes Haemagglutinin-Neuraminidase (HN) protein which makes the virus bind to a host, and Fusion (F) protein which makes the envelope fuse with the host cell. F and HN proteins are glycoprotein and distributed on the surface of viral envelope.
Depending on the phylogenetic analysis of the F protein in Newcastle disease virus, the Newcastle disease virus can be classified into genotype I to genotype VIII. Genotypes VI and VII are reported in Asia including Indonesia, China, Taiwan, and Japan. The genotype VII virus is classified into sub-genotypes VIIa and VIIb which were reported in Asia and Africa, respectively (Herczeg et al., 1999). In Korea, genotype VIIa virus was reported, and linear epitopes of the HN proteins have the same amino acid sequence as those of vaccine virus (Kwon et al., 2003; Lee et al., 2004). A part of Chinese genotype VII viruses cannot be prevented by conventional vaccine completely, but the reason was not reported (Yu et al., 2001).
F protein belongs to type I membrane glycoprotein and includes trimeric structure (Gorman et al., 1988; Russell et al., 1994; Reitter et al., 1995). HN protein belongs to type II membrane glycoprotein and includes tetramer on the surface of virus envelope, so as to invade into the cell membrane (Gorman et al., 1988; Ng et al., 1989). F protein has thirteen (13) cysteine residues and five (5) glycosylation sites, and HN protein has fourteen (14) cysteine residues and four (4) glycosylation sites.
Cysteine residue in protein plays an important role in protein conformation and its oligomerization. In addition, the cysteine residue has an influence on epitope formation and the glycosylation site of a peptide. Mutation on cysteine residue, C123 of HN protein does not form the disulfide bond between molecules, and covalent dimer. In addition, the mutation on cysteine residue affects a structure of antigen (McGinnes and Morrison, 1994, 1997). All Newcastle disease viruses have well-conserved cysteine residues, and it means a strict interaction between the structure and function. Biological function of each cysteine residue in F protein have not been revealed. Only C199 and C76 form a disulfide bond which maintains the linkage between F1 and F2 polypeptides after cleavage of F protein (Wang et al., 1992).
F and HN proteins are important protective antigens, and their epitopes were determined by using mouse monoclonal antibody. Until now, three conformational epitopes in F protein and two conformational epitopes in HN protein, and a linear epitope were reported (Nishikawa et al., 1983, 1986; Sakaguchi et al., 1989). Eight amino acids constituting a linear epitope of HN protein are 346Aspartic acid-Glutamic acid-Glutamine-Aspartic acid-Tyrosine-Glutamine-Isoleucine-Arginine353.
Because F and HN proteins are important protective antigens of Newcastle disease virus, an infection and death caused by Newcastle disease virus can be prevented by immunizing fowls with F and HN proteins as antigens. In addition, the avian serum test can be carried out by using the antibodies against F and HN proteins. Particularly, the antibody against HN protein is used all over the world in hemagglutination-inhibition test. The hemagglutination-inhibition test is preferred because of high relationship between titer of antibody and disease protection, but has a disadvantage in variation of antibody titer, and difficult automation compared to the ELISA test. ELISA is advantageous in automation and measuring the reaction result as a numerical value. However, because ELISA uses all virus antigens, more antibodies to abundant nucleocapsid protein lowers the correlation with antibody titer obtained by the hemagglutination method.